The DNA replication of the small Escherichia coli plasmids Colicin El (Col El), pl5A and derivatives of the R plasmid, Rl00.l, is being studied. The specific aims of this project are (1) to define the origin of replication of Colicin El and similar plasmids such as pl5A, (2) to test known E. coli DNA replication genes for their requirement in Col El DNA synthesis, (3) to isolate mutants of E. coli which fail to replicate these plasmids under non-permissive conditions to define further the E. coli functions required for plasmid DNA replication, (4) to select mutants of Col El which replicate more or less frequently than the wild type, (5) to select mutants of smaller derivatives of the R plasmid, Rl00.l, which cannot replicate their DNA and (6) to develop an in vitro DNA synthesizing system for the study of Rl00.l, and its mutants. The overall objective is to understand the positive and/or negative control mechanisms which operate to control the initiation of DNA replication and to establish an in vitro system where these mechanisms can be demonstrated.